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Back to 2005 Research
Clare Waterman-Storer
Microscopes and Migration 2005
Cell migration relies on a cycle of four spatiotemporally coordinated molecular processes: actin polymerization-driven protrusion of the plasma membrane at the leading edge; acto-myosin dependent formation of new focal adhesion sites to the extracellular matrix under the newly spread cell region; disruption of older adhesion sites at the cell rear; and contraction of the actin cytoskeleton by myosin-based forces balanced between the stronger cytoskeleton-substratum coupling in the front and the weaker coupling at the rear yielding net movement of the cell body. Thus, spatiotemporal control of f-actin polymerization and actomyosin contraction coupled to substrate adhesion is essential to generate the self-perpetuating asymmetry consisting of lamellipodial protrusion in the cell front and retraction in the cell rear to drive motility. It is thought that MTs provide spatial and temporal orchestration of these f-actin-based protrusive, contractile, and adhesive events via either structural linkage to actin to direct is organization or by providing spatiotemporal regulation of signals that control actin activities.
The overall theme of our section of the 2005 Physiology course was to study the relation between cell migration and adhesion dynamics in migrating cells using state-of-the-art digital light microscopy.
Four student groups in our session addressed the following questions:
What is the relationship between substrate adhesion and contractility in keratocyte motility?
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Figure 1: Migrating fish skin keratocytes seen by immunofluorescence showing the actin cytoskeleton (blue) and vinculin, a focal adhesion protein (red)
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One group of students used fish skin keratocytes as a model system for a very simple motile cell to analyze how substrate adhesion and contractility of the actin cytoskeleton are related (Fig 1). The students prepared cultures of fish skin keratocytes on different concentrations of fibronectin, an extracellular matrix protein, and treated the cells with actin and myosin inhibitors. They then observed the cells by low magnification phase contrast on a microscope equipped with an automated stage so that multiple positions could be recorded over time. The students analyzed a large number of cells and found that exhibit a biphasic response to increasing adhesion or decreasing contraction, with maximal migration speed at intermediate adhesion sterength and intermediate contraction inhibition, indicating a complex interplay between these parameters.
How does myosin activity organize actin and focal adhesions to the substrate?
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Figure 2: Images of EGFP-tagged paxillin during washout of myosin inhibitors
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Focal adhesions are the sites that connect the actin cytoskeleton in cells to its underlying substrate. It is thought that myosin-II dependent contractility in the actin cytoskeleton, which is linked to integrins in the plasma membrane, induces the clustering of integrins. This tension may increase the affinity of integrins for the extracellular matrix and initiate the formation of focal adhesions. When contractility is inhibited, both the contractile actin bundles as well as the focal adhesions disassemble. One group of students took up the very difficult task to observe changes in focal adhesion dynamics, when contractility is induced in cells. They did this by incubating mouse embryonic fibroblasts expressing fluorescently labeled focal adhesion proteins in a cocktail of myosin inhibitors. They then mounted the cover glasses with cells in a perfusion chamber and induced contractility by washing away the myosin inhibitors while observing focal adhesions by total internal reflection microscopy. This is a technically challenging experiment, but the students manged to obtain several striking time-lapse series. One clear result was that the fluorescence intensity of individual focal adhesions increased dramatically during the drug washout indicating clearly that proteins are recruited from the cytoplasmic pool and not only from coalescing small adhesions (Figure 2).
What is the relationship between contractility and protein affinity to adhesions sites in migrating cells?
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Figure 3: FRAP of GFP integrin in a focal adhesion.
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Another group studied the dynamics of different focal adhesion proteins by FRAP (fluorescence recovery after photobleaching). The students expressed different EGFP-tagged focal adhesion proteins in mouse embryonic fibroblasts and bleached selected spots in focal adhesions with a focused laser beam on a Deltavision RT microscope system (Applied Precision). The rate by which fluorescence recovers in the bleached area gives some idea about how freely molecules can bind to and dissociate from a certain structure. One surprising result was that different proteins displayed widely different turnover dynamics. For example, they found that vinculin was very tightly bound to adhesions, while talin was much more loosely bound. This is surprising, considering that both proteins are thought to mediate a structural link between actin and integrins. They then tested the effects of reducing contraction on the affinity of proteins for focal adhesions. They found that reduction of contraction either by pharmacological inhibition of myosin activity caused a remarkable decreas in vinculin affinity for foca;l adhesions, with other adhesion proteins largely unaffected. This suggests that vinculin may be a tension-sensitive regulator of adhesion strength.
Do the lamella and the lamellipodium share the same or different actin pools?
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Figure 4: Photoactivation of GFP-actin in a pattern of stripes reveals the spatial variation in actin turnover in an epithelial cell.
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Using fluorescent speckle microscopy of F-actin in migrating epithelial cells, we have shown that two distinct F-actin networks exist at the leading edge, the lamellipodium and the lamella. The last group of students used photoactivatable GFP to test the hypothesis whether actin can freely redistribute between the lamella and the lamellipodium. This GFP variant is not fluorescent, but becomes fluorescent when it is illuminated with UV light. The students used a very new illumination system (Mosaic, Photonic Instruments) to do these experiments, which allowed them to photoactivate any region of interest in a cell. They used PtK1 epithelial cells that expressed photoactivatable GFP-actin and constitutively active Rac1, which ensures a pronounced lamellipodium all around the edge of the cell (Figure 4). It was very clear in these experiments that photoactivated actin becomes incorporated into the newly polymerizing lamellipodial actin meshwork rapidly. However, it was difficult to reach quantitative conclusions of whether this redistribution can be attributed to diffusion alone. An unexpected side-result was that the photoactivatable GFP-actin induced actin bundling in cells.
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